Growth and disorder of macromolecular crystals: insights from atomic force microscopy and X-ray diffraction studies
Introduction
A detailed understanding of the function of proteins, complexes, and viruses requires knowledge of their three-dimensional structure. Despite enormous progress over the last decade in macromolecular production, in automated screening, and in X-ray data collection and analysis, the rate at which new structures are obtained by X-ray crystallography is still largely limited by the difficulty of obtaining high-quality crystals and of maintaining their quality throughout the data collection process [1], [2], [3]. In a perfect crystal, molecules would be packed in a perfectly periodic arrangement and each molecule would be identical. Macromolecular crystals can exhibit a variety of imperfections or disorder that limit the accuracy of molecular structure determinations and often make structure determination impossible. Consequently, the most important objective of fundamental studies of macromolecular crystals and their growth is to identify and reduce this disorder [1], [2], [3], [4], [5], [6].
To achieve this objective, we must address basic questions such as: (1) How do macromolecular crystals nucleate and grow? (2) How do particular kinds of disorder affect the X-ray diffraction properties? (3) What kinds of disorder do macromolecular crystals exhibit? (4) What properties of the molecules and their interactions are most relevant in producing disorder? (5) What role do impurities and other kinds of molecular heterogeneity present in growth solutions play? (6) How does disorder arise during growth, and during post-growth handling including soaks, flash cooling, and irradiation? and (7) How can this disorder be reduced or eliminated by proper choice of expression, purification, growth, and handling methods? These questions parallel those that materials scientists have grappled with for more than a half-century in their quest to develop high-quality inorganic single crystals for the electronics and optical communications industries [7].
Over the last 15 years, a number of powerful experimental techniques have been applied to study macromolecular crystals and their disorder. In this chapter, we focus on atomic force microscopy (AFM) and on X-ray diffraction and imaging techniques. These techniques can be applied in situ and are highly complementary. Atomic force microscopy provides spectacular real-space resolution and can directly image growth processes, individual molecules, and defects at crystal surfaces. X-ray imaging and diffraction provide excellent reciprocal space resolution and can probe mosaicity, strain, and bulk crystal defects over the full temperature range of interest in crystallography. These techniques have allowed us to make substantial headway in addressing the above questions, with important implications for the general practice of macromolecular crystal growth and crystallography.
We begin in Section 2.1 with a discussion of atomic force microscopy, emphasizing factors important to high-resolution in situ studies of macromolecular crystals. Section 2.2 describes X-ray imaging and high-resolution diffraction techniques. Section 3 shows how AFM has been used to obtain a very detailed and quantitative understanding of how macromolecule and virus crystals grow. In the remainder of the article, we focus on imperfections or disorder in macromolecular crystals. Section 4 gives a review of X-ray diffraction measures of macromolecular crystal quality of relevance to structure determinations and discusses the kinds of disorder that may contribute to each. Section 5 describes applications of AFM and X-ray diffraction techniques to disorder created during growth, by seeding, by impurities, by post-growth treatments, and by flash cooling. Throughout this article we will attempt to relate fundamental insights to practical implications and, where possible, to methods for obtaining higher quality crystals. With a little experience AFM and X-ray imaging can quickly provide information not readily obtained using standard biochemical and crystallographic methods. Consequently, we anticipate that they will become an integral part of the toolkit used to address challenging crystals and structures.
Section snippets
Atomic force microscopy
Because of the large diameters of macromolecules and viruses, atomic force microscopy (AFM) allows surfaces of growing macromolecular crystals to be imaged in situ with molecular resolution. It provides information about growth and perfection of macromolecular crystals that cannot be obtained by other physical and analytical techniques. In addition to the visualization of crystal surface morphologies, growth mechanisms, impurity effects, and defect formation, high-resolution AFM images can
Growth mechanisms
AFM studies of macromolecular crystallization [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49] show that the surfaces of macromolecular crystals are smooth and growth proceeds layer-by-layer, with the sources of the growth layers being either two-dimensional (2D) nuclei or screw dislocations. In 2D nucleation, islands
X-ray diffraction measures of macromolecular crystal disorder
We now turn to discuss imperfections or disorder in macromolecular crystals. Macromolecular crystallographers use three primary measures to characterize crystal quality: the diffraction resolution, the B or temperature factor, and the mosaicity [2], [59]. We begin by addressing the question: How are these parameters related to crystal disorder?
Disorder in macromolecular crystals: Character, origin, and remedies
The combination of atomic force microscopy and X-ray diffraction techniques described in Section 2 has provided detailed insight into the nature and origin of disorder in macromolecule and virus crystals. As we emphasize below, these insights have several practical implications for how these crystals can be improved.
Concluding remarks
Atomic force microscopy, X-ray topography, and high-resolution reciprocal space scanning have provided extremely detailed insight into macromolecular crystal growth and disorder from the crystal to molecular scales. The growth mechanisms have been identified and quantified, and many important kinds of disorder and their sources characterized. These results have broad implications for macromolecular crystal growth and crystallography. Until recently a significant barrier between communities has
Acknowledgements
Research summarized here was supported by grants from the National Aeronautics and Space Administration (NAG8-1357, NAG8-1408, NAG8-1569, NAG8-1574, and NAG8-1831) and the National Institutes of Health. Part of this work was performed under the auspices of the US Department of the Energy by the University of California, Lawrence Livermore National Laboratory under Contract W-7405-Eng-48. The authors wish to thank A. McPherson, M. Plomp, and Yu.G. Kuznetsov at UC Irvine and I. Dobrianov, C.
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